To compare the cellular mechanism of protection against drug-stimulated coronary vasospasm by multi-week estrogen plus progesterone (P) versus medroxyprogesterone acetate (MPA) treatments, we studied intracellular Ca2+ and protein kinase C (PKC) signals. Ovariectomized monkeys were treated by slow release silastic implants with either P or MPA for 4 weeks added to estradiol-17 (E2) begun 2 weeks earlier. A third group received E2 for 2 weeks, and withdrawal (W) of E2 (no steroid treatment) during the last 4 weeks. Using monkey coronary artery vascular muscle cells (VMC) in primary culture conditions that maintain contractile function, we examined potential mechanisms of coronary artery hyperreactivity. Intracellular Ca2+ and PKC were labeled by the fluorescent indicators fluo3 and hypericin, respectively, and analyzed in single VMC by a high sensitivity photon counting camera system. In response to stimulation with 10 fM serotonin and 100 nM U46619 (thromboxane A2 mimetic), VMC isolated from 5 P treated monkeys showed a transient Ca2+ increase for 2-5 min and no significant increase in PKC activation, while VMC from 6 MPA treated or 7 W monkeys showed marked increases of both Ca2+ and PKC signals that persisted for >30 min, while Ca2+ activated increased locally by up to 20X, and increases averaged 30-40% over whole VMC. P versus MPA or W differences in VMC Ca2+ and PKC signals were maintained up to 14 days after being isolated. Therefore, the interference by MPA with the beneficial effect of E2 to reduce constrictor reactivity persisted in culture for at least 14 days. We hypothesize that greatly prolonged high Ca2+ levels found during and after stimulation of VMC from MPA treated or W monkeys result from accelerated and prolonged release of Ca2+ from central stores, with concomitant changes in PKC. We conclude that 4 week treatment with MPA, a synthetic progestin negates the protective effect of E2 on coronary reactivity and allows increased mobilization of Ca2+ and PKC, resulting in prolongation of intense contraction, and thus the likelihood of vasospasm and ischemia. In contrast, natural P treatment does not interfere with protection against coronary vasospasm.